Rice-origin gibberellin 2beta-hydroxylase genes and utilization thereof

ABSTRACT

Novel GA2β-hydroxylase genes were successfully isolated from rice. In addition, plants whose plant type has been modified compared with their wild type counterparts, were successfully constructed via these genes.

Technical Field

[0001] The present invention relates to genes of rice involved ingibberellin biosynthesis and uses thereof.

Background Art

[0002] Gibberellins (GAs) form a large family of tetracyclic diterpenoidcarboxylic acids that have the basic structure called ent-gibberellane(FIG. 1A). They control multiple processes in the life cycle of higherplants, which are essential for normal plant growth and development(Graebe, J. E. (1987). Ann. Rev. Plant Physiol., 38, 419-465; Hooley, R.(1994). Plant Mol. Biol., 26, 1529-1555). Biologically active GAs, suchas GA₁, are produced from trans-geranylgeranyl diphosphate mediated bythe sequential actions of cyclases in the plastids, membrane-associatedmonooxygenases at the endoplasmic reticulum and soluble2-oxoglutarate-dependent dioxygenases located within the cytosol(reviewed in Hedden, P. and Kamiya, Y. (1997). Ann. Rev. Plant Physiol.Plant Mol. Biol., 48, 431-460; Lange, T. (1998). Planta, 204, 409-419).The biosynthetic pathway of GA is well established (FIG. 1B). The2-oxoglutarate-dependent dioxygenases catalyze the later steps in thebiosynthetic pathway, including the removal of C-20 by GA 20-oxidase andthe introduction of the 3β-hydroxyl group by GA 3β-hydroxylase tosynthesize biologically active GAs. A third dioxygenase, GA2β-hydroxylase, introduces a 2β-hydroxyl group resulting in biologicallyinactive GAs that cannot be converted into active forms.

[0003] In recent years, cDNAs and genomic clones encoding GAbiosynthetic enzymes have been isolated from various plant species(reviewed in Hedden, P. and Kamiya, Y. (1997). Ann. Rev. Plant Physiol.Plant Mol. Biol., 48, 431-460; Lange, T. (1998). Planta, 204, 409-419).Availability of these clones has been clarifying the regulation of GAbiosynthesis. The GA 20-oxidases, for example, are shown to be encodedby several genes that are differentially regulated throughout plantdevelopment (Phillips, A. L. et al. (1995). Plant Physiol., 108,1049-1057; Garcia-Martinez, J. L. et al. (1997). Plant Mol. Biol., 33,1073-1084). Although GA 2β-hydroxylases play an important role indetermining the endogenous concentration of bioactive GAs, the genes forthese enzymes have not been isolated until recently. The first isolationof GA 2β-hydroxylase genes was from scarlet runner bean (Phaseoluscoccineus L.) and Arabidopsis thaliana by a functional screening method(Thomas, S. G. et al. (1999). Proc. Natl. Acad. Sci. USA, 96,4698-4703).

[0004] GAs are involved in many developmental processes, includinggermination, stem elongation, flowering, and fruit development.Therefore, modifications of these processes by application of chemicalsthat alter GA content are common agronomic and horticultural practices.For instance, GA3 is used to stimulate berry growth in seedless grapeproduction (Christadoulou, A. J. et al. (1968) Proc. Am. Soc. Hort.Sci., 92, 301-310), and GA biosynthesis inhibitors are used as growthretardants to control the height of cereal crops and ornamental plants(Hedden, P. and Hoad, G. (1994). New York: Marcel Dekker, pp. 173-198).An alternative approach to the exogenous application of chemicals wouldbe to modify the endogenous content of GAs via genetic manipulation oftheir biosynthesis. The recent cloning of several genes involved in GAbiosynthesis provided the means to test the feasibility of thisapproach. Isolation of genes encoding GA 2β-hydroxylase was, inparticular, expected to bring a powerful tool to control the bioactiveGA content in transgenic plants.

[0005] A number of GA-responsive mutants have been isolated from variousplant species, such as maize, pea, tomato, Arabidopsis, and rice(Phinney, B. 0. (1956). Proc. Natl. Acad. Sci. USA, 42, 185-189;Koornneef, M. (1978). Arabidopsis Ins. Serv., 15, 17-20; Koornneef, M.et al. (1990). Theor. Appl. Genet., 80, 852-857; Reid, J. B. and Ross,J. J. (1993). Int. J. Plant Sci., 154, 22-34; Murakami, Y. (1972). PlantGrowth Substances 1970. (Carr, D. J. ed.) Berlin: Splinger-Verlag, pp.166-174). Phenotypes resulting from reduced GA production in spontaneousmutants of Arabidopsis imply the role of GAs in stem elongation andflowering (Koornneef, M. and van der Veen, J. H. (1980). Theor. Appl.Genet., 58, 257-263; Sponsel, V. M. et al. (1997). Plant Physiol., 115,1009-1020). GAl encodes copalyl-diphosphate synthase (CPS). Nullmutations in this locus inhibit stem elongation in long day conditionsto cause flowering without bolting and both stem elongation andflowering in short day conditions (Wilson, R. N. et al. (1992). PlantPhysiol., 100, 403-408; Sun, T. -P. and Kamiya, Y. (1994). Plant Cell,6, 1509-1518). GA4 encodes GA 3β-hydroxylase (Chiang, H. -H. et al.(1995). Plant Cell, 7, 195-201; Williams, J. et al. (1998). PlantPhysiol., 117, 559-563), while GA5 and GA6 encode distinct GA20-oxidases (Xu, Y. L. et al. (1995). Proc. Natl. Acad. Sci. USA., 92,6640-6644; Sponsel, V. M. et al. (1997). Plant Physiol., 115,1009-1020). Null mutations in both GA4 and GA5 result in semi-dwarfswith normal flower development. In contrast, loss of function of GA6results in short inflorescences, reduced fertility and short siliques(Sponsel, V. M. et al. (1997). Plant Physiol., 115, 1009-1020).

[0006] GA-deficient mutants have also been isolated from rice (dx: d35and dy: d18). The rice dwarf mutants have considerable agriculturalsignificance. For example, sd-1 mutants are especially important forrice breeding because they are the genetic basis of high yielding,semi-dwarf varieties. Rice d18 mutants are GA responsive dwarf, andmultiple alleles have been identified; Housetu-waisei (d18^(h)),Akibare-waisei (d18-AD), Kotake- tamanishiki (d18^(k)), and Waito-C(d18-w) were isolated from different parental ecotypes. The analyses ofGA intermediates in d18 mutants demonstrated that the conversion to3β-hydroxyl GAs was blocked in these mutants. This resulted in theaccumulation of endogenous level of GA₂₀ and the drastic decrease ofbioactive GA₁ content (Kobayashi, M. et al. (1988). Plant Cell Physiol.,30(7): 963-969; Kobayashi, M. et al. (1994). Plant Physiol. 106:1367-1372; Choi Y-H. et al., (1995). Plant Cell Physiol. 36(6):997-1001). These findings strongly suggest that the D18 gene encodes aGA 3β-hydroxylase and reduction of GA₁ suppresses stem elongation inmutant plants.

[0007] The dwarf stature characteristic is one of the most valuabletraits for breeding of agricultural and horticultural crops includingfruit trees because this feature enables high density planting,efficient reception of light, decrease of wind damage, and reduction offarming labor. It is possible to reduce endogenous levels of bioactiveGAs in transgenic plants. For example, antisense expression ofArabidopsis GA 20-oxidase gene and tobacco GA 3β-hydroxylase genedecreases the level of active GAs, and results in semi-dwarf phenotypes(Coles et al., (1999). Plant. J. 17, 547-556; Itoh et al., (1999).Plant. J. 20, 15-24). However, this method of producing dwarf plants byantisense expression of these active GA-forming enzyme genes has twomajor defects: 1) it is difficult to predict and regulate an endogenouslevel of GA, because expression of homologue genes which exist in thesame species as the plant, into which the antisense construct will beintroduced, may not be suppressed and the half-life of activegibberellins is extended due to the suppression of expression of genesencoding 2β-hydroxydases that produces biologically inactive GAs, and 2)it is necessary to isolate the corresponding cDNA from the same plantspecies as the plant into which the antisense construct will beintroduced.

[0008] In contrast, since the structure of active GAs, which aresubstrates for GA deactivation enzymes, is preserved in other plants,overexpression of GA deactivation enzyme genes, such as GA2β-hydroxylase gene, is probably effective in heterologous plantspecies. Moreover, it could easily regulate the active GA content to apreferable level via modification of transgene expression.

Disclosure of the Invention

[0009] The present invention has been made in view of the advantage oftargeting GA deactivation enzymes for controlling the GA content inplants. A primary objective of this invention is to provide a novel GA2β-hydroxylase gene originating in a plant, especially in rice plant.Another objective of this invention is to modify the plant type of aplant by controlling the plant GA content utilizing this gene.

[0010] The major metabolic pathway of GAs is initiated by2β-hydroxylation, a reaction catalyzed by a soluble2-oxoglutarate-dependent dioxygenase. For the production of a plant,whose plant type is modified by controlling the GA content, the presentinventors have isolated a GA 2β-hydroxylase gene from rice andcharacterized said gene.

[0011] First, to isolate GA 2β-hydroxylase genes originating in rice,degenerate primers were designed based on a comparison of putative aminoacid sequences encoded by the rice GA 2β-hydroxylase gene, Marahmacrocarpus mRNA for dioxygenase, rice GA 20-oxidase gene, two distinctrice GA 3β-hydroxylase genes, and other 2-oxoglutarate-dependentdioxygenase genes. PCR was performed using the primers and , the genomicDNA from rice (as a template) to isolate a plurality of independentclones. Of these clones, one clone presumed to encode GA 2β-hydroxylasewas selected, and its sequence information was used to search databasesin order to obtain a rice EST clone. Then, utilizing primers designedbased on the sequence information of this EST clone, PCR was carried outusing the rice genomic DNA as a template. Furthermore, using thePCR-amplified fragments thus obtained as probes, a rice cDNA library andgenomic DNA library were screened. This resulted in successful isolationof the genomic DNA and cDNA encoding the rice GA 2β-hydroxylase (theclone encoding the rice GA 2β-hydroxylase was designated “OsGA2ox1 ”).

[0012] Then, the present inventors examined the activity of recombinantproteins obtained by expressing the OsGA2ox1 cDNA in E. coli, confirmingthat these recombinant proteins have the GA 2β-hydroxylase activity toconvert C₁₉-GAs and C₂₀-GAs into their corresponding 2β-hydroxylatedproducts.

[0013] Analysis of OsGA2ox1 expression in various parts of rice revealedits localization in the basal regions of a differentiated leafprimordia, epithelium, and aleurone layer.

[0014] Furthermore, the present inventors produced transgenic riceplants expressing the OsGA2ox1 cDNAs in order to establish that theseplants become dwarf compared to control plants.

[0015] As described above, the present inventors have succeeded inisolating novel GA 2β-hydroxylase genes from rice, and utilizing thesegenes by producing plants whose plant type has been modified as comparedwith the wild type plants.

[0016] Therefore, the present invention relates to a novel GA2β-hydroxylase gene originating in rice, and then use of the gene,particularly, to produce plants whose plant type has been modified. Morespecifically, the invention provides:

[0017] (1) a DNA encoding a protein having gibberellin 2β-hydroxylaseactivity, selected from the group consisting of:

[0018] (a) a DNA encoding a protein comprising the amino acid sequenceset forth in SEQ ID NO: 1,

[0019] (b) a DNA containing a coding region of the nucleotide sequenceset forth in SEQ ID NO: 2, and

[0020] (c) a DNA encoding a protein comprising the amino acid sequenceset forth in SEQ ID NO: 1, wherein one or more amino acid residues aresubstituted, deleted, added, and/or inserted;

[0021] (2) a DNA according to (1), which is used for producing dwarfedplants;

[0022] (3) a DNA for suppressing the expression of endogenous DNAaccording to (1) within plant cells, selected from the group consistingof:

[0023] (a) a DNA encoding an antisense RNA complementary to the DNAaccording to (1) or its transcription product,

[0024] (b) a DNA encoding an RNA having the ribozyme activity tospecifically cleave the transcription product of the DNA according to(1), and

[0025] (c) a DNA encoding an RNA that suppresses the expression of theendogenous DNA according to (1) by co-suppression, wherein said DNA has70% or more homology to a DNA comprising the nucleotide sequence setforth in SEQ ID NO: 2;

[0026] (4) a vector harboring the DNA according to any one of (1)through (3);

[0027] (5) a transformed plant cell harboring the DNA according to anyone of (1) through (3) in an expressible state;

[0028] (6) a transgenic plant containing the transformed plant cellaccording to (5);

[0029] (7) a propagative material of the transgenic plant according to(6);

[0030] (8) a protein encoded by the DNA according to (1);

[0031] (9) a method for producing the protein according to (8), whereinsaid method comprises culturing the transformed cells harboring the DNAaccording to (1) in an expressible state and recovering the expressedprotein from said cells or the culture supernatant thereof;

[0032] (10) a method for modifying the plant growth, wherein said methodcomprises controlling the expression level of the DNA according to (1)in plant cells;

[0033] (11) a method for modifying a plant type, wherein said methodcomprises controlling the expression level of the DNA according to (1)in plant cells; and

[0034] (12) a method according to (10) or (11), wherein the DNAaccording to any one of (1) through (3) is expressed in plant cells.

[0035] The present invention provides a novel GA 2β-hydroxylase isolatedfrom rice and a DNA encoding this enzyme. The nucleotide sequence of theOsGA2ox1 cDNA isolated by the present inventors and included in the DNAsof this invention, and the amino acid sequence of the OsGA2ox1 proteinare set forth in SEQ ID NOs: 2 and 1, respectively.

[0036] The OsGA2ox1 cDNA contains an open reading frame of 1,146 bpencoding a protein consisting of 382 amino acid residues. The proteinencoded by the cDNA has the amino acid sequence that is conserved withinthe dioxygenases involved in GA biosynthesis (cf. FIG. 2A), retains theamino acid residues that bind to Fe at their active sites, and alsoshows a significant sequence homology with those of other GA2β-hydroxylases. Furthermore, the protein encoded by the cDNA hasactivity to produce 2β-hydroxylated products from a wide range ofC19-GAs. Therefore, the OsGA2ox1 cDNA isolated by the present inventorsis assumed to encode GA 2β-hydroxylase.

[0037] GA 2β-hydroxylases directly regulates levels of bioactive3β-hydroxylated GAs such as GA₁, and GA₄, as well as levels of theirimmediate precursors such as GA₂₀ and GA₉ to produce bio-inactive2β-hydroxylated GAs. In fact, recombinant proteins encoded by theOsGA2ox1 cDNA isolated by the present inventors have the activity toconvert a wide variety of C19-GAs into the corresponding 2β-hydroxylatedproducts. Therefore, GA 2β-hydroxylase of the present invention and DNAencoding the enzyme may be useful in manufacturing bio-inactive GAs.

[0038] Furthermore, studies on GA-deficient mutants and actions ofexogenous GAs and/or inhibitors applied to plants for GA biosynthesishave revealed that GAs are essential and potent regulators for plantgrowth. These GAs influence various phenomena in the growth of plantshaving a relatively high stature, and are also involved in thestimulation of stem elongation. In fact, plants in which the OsGA2ox1cDNA of the present invention is expressed become severely dwarfed.Therefore, GA 2β-hydroxylase of the present invention and DNA encodingthe enzyme may be useful in modifying plant growth, for example,production of a plant whose plant type differs from that of a wild type.Modification of plant type, dwarfing in particular, provides a varietyof agronomical advantages such as a high density of planting, efficientphotoreception, decrease in wind damage, reduction of farming labor,etc. Dwarfing is thus the most valuable trait for breeding agriculturaland horticultural products, including fruit trees.

[0039] The GA 2β-hydroxylase of this invention can be prepared as arecombinant protein via methods known to those skilled in the art or asa natural protein. A recombinant protein can be prepared, as describedbelow, for example, by inserting DNA (e.g., SEQ ID NO: 2) encoding theGA 2β-hydroxylase of this invention into an appropriate expressionvector and purifying the protein from cells transformed with the vector.A natural protein can be prepared, for example, by immunizing suitableanimals with the prepared recombinant protein or its partial peptide,binding the thus prepared antibody to a column for affinitychromatography, contacting the column with extracts prepared fromtissues of rice expressing the protein of this invention, and purifyingthe protein binding to the column.

[0040] The GA 2β-hydroxylase of this invention includes wild typeproteins (SEQ ID NO: 1) in which partial amino acid residues aremodified, while retaining the function of the wild type proteins. Anexample of the method for preparing such modified proteins well known tothose skilled in the art include the site-directed mutagenesis method(Kramer, W. and Fritz, H.-J. Methods in Enzymology, 154: 350-367, 1987).Amino acid mutations may also occur spontaneously. The GA 2β-hydroxylaseof this invention thus include proteins that retain the GA2β-hydroxylase activity of the wild-type protein and those that aremodified via substitution, deletion, addition, and/or insertion of oneor more amino acid residues in the amino acid sequence of the wild typeprotein. There is no particular limitation on the site and number ofsuch amino acid modifications in the protein so far as the modifiedprotein retains the GA 2β-hydroxylase activity. The number of amino acidthat can be modified may be usually not more than 50 amino acidresidues, preferably not more than 30, more preferably not more than 10,and most preferably not more than 3 amino acid residues.

[0041] The term “GA 2β-hydroxylase activity” used herein refers to theactivity to synthesize 2β-hydroxylated products of the substrate,C₁₉-GAs (e.g., GA₁, GA₄, GA₉s, or GA₂₀). The activity can be detected asfollows. In general, cDNA obtained is inserted into an expression vectorand overexpressed as a fusion protein in E. coli. Using the resultingcell extract as an enzyme solution and C₁₉-GAs as a reaction substrate,the reaction is performed in vitro in the presence of the co-factors,ferrous ion and 2-oxoglutarate. Finally the reaction product(2β-hydroxylated product) is confirmed by GC-MS.

[0042] The present invention also provides a DNA encoding the GA2β-hydroxylase of this invention. This DNA includes both cDNA andgenomic DNA as long as both encode GA 2β-hydroxylase of the presentinvention. cDNAs encoding the OsGA2Ox1 proteins can be prepared, forexample, by performing RT-PCR using primers designed based on theinformation of the nucleotide sequence set forth in SEQ ID NO: 2 and, asa template, total RNA isolated from rice plants (e.g. total RNA derivedfrom inflorescence). The genomic DNA can be prepared, for example, byperforming PCR using primers designed based on the information of thenucleotide sequence set forth in SEQ ID NO: 2 and, as a template, thegenomic DNA of rice.

[0043] DNAs encoding GA 2β-hydroxylase of the present invention can beused, for example, for producing recombinant proteins. Recombinantproteins can be produced, as described below. First, a full-length cDNAis synthesized by RT-PCR using primers provided with restriction enzymesites and subcloned into multi-cloning sites of the pMAL-c2 expressionvector (NEB). This construct is used to transform Escherichia colistrain BL21 cells (protease-deficient strain) by standard methods. Usingthe transformant thus obtained, the protein is induced. E. coli arecultured (by shaking) in a 2×YT medium containing 0.2% glucose at 37° C.When an OD₆₀₀ value reaches around 0.6, IPTG is added to a finalconcentration of 1 mM, and culturing is further continued at 18° C. for24 h. Extraction of an enzyme solution is performed as follows. Afterculturing, cells are collected and lysed in a suspension buffer (50 mMTris-HCl (pH 8.0) containing 10% glycerol, 2 mM DTT, and 1 mg/mllysozyme). The cell suspension is allowed to stand at 4° C. for 30 min,and then incubated at −80° C. until it becomes completely frozen. Thefrozen suspension is thawed and sonicated for 30 s twice at 5-minintervals at the MAX level with the Sonicator (Heat Systems-Ultrasonics,Inc., Model W-225R). The suspension thus treated is centrifuged (at15,000 rpm and 4° C. for 20 min), and the supernatant is used as a crudeenzyme solution.

[0044] Furthermore, preparation of the purified protein can be carriedout, by expressing the GA 2β-hydroxylase of this invention in E. coli(or the like) as a fusion protein with the histidine tag,maltose-binding protein, or glutathione-S-transferase (GST), andsubsequently purifying them on a nickel column, an amylose-column, or aGST-glutathione column, respectively. Then, after the purification, theabove-described tags can be cleaved off using limited proteases, suchas, thrombin and factor Xa as required.

[0045] As described above, the genes isolated by the present inventorsare assumed to be involved in the plant growth through the production ofbiologically inactive GAs. Therefore, plant growth may be controlled byregulating the expression of these genes. Since these genes inparticular are thought to be involved in the internodal growth ofplants, this gene may be utilized in the control of plant stature.Control of plant stature provides a variety of industrial advantages.For example, the shortened stature caused by increasing the expressionof the gene of this invention in a plant can make the plant resistant tobending thereby increasing the fruit weight. Furthermore, the shortenedstature makes the size of the plant per stub more compact so that thenumber of plants to be planted per unit area can be increased. Thisdense planting is highly important in the production of agriculturalproducts including rice, wheat, maize, etc., in particular. DNA encodingthe GA 2β-hydroxylase of the present invention may be applicable todwarf flowering plants, dwarf fruit trees, etc.

[0046] On the other hand, the yield of plants as a whole may be enhancedby lengthening plant stature through the repressed expression of genesof this invention within the plants. This is useful for improving, forexample, feed crop yields as a whole.

[0047] In the present invention, a variety of methods known to thoseskilled in the art are available for suppressing the expression of genesof this invention to control plant growth. Herein, “suppression ofexpression of genes” includes suppressions of both gene transcriptionand translation into proteins, and includes not only completesuppression but also decrease in the gene expression.

[0048] The expression of a specific endogenous gene in plants can besuppressed by conventional methods utilizing antisense technology. Eckeret al. were the first to demonstrate the effect of an antisense RNAintroduced by electroporation in plant cells by using the transient geneexpression method (Ecker, J. R. and Davis, R. W. (1986). Proc. Natl.Acad. Sci. USA 83, 5372). Thereafter, the target gene expression wasreportedly reduced in tobacco and petunias by expressing antisense RNAs(van der Krol, A. R. et al. (1988). Nature 333, 866). The antisensetechnique has now been established as a means to suppress target geneexpression in plants.

[0049] Multiple factors cause antisense nucleic acid to suppress thetarget gene expression. These include inhibition of transcriptioninitiation by triple strand formation; suppression of transcription byhybrid formation at the site where the RNA polymerase has formed a localopen loop structure; transcription inhibition by hybridization with theRNA being synthesized; suppression of splicing by hybrid formation atthe junction between an intron and an exon; suppression of splicing byhybrid formation at the site of spliceosome formation; suppression ofmRNA translocation from the nucleus to the cytoplasm by hybridizationwith mRNA; suppression of splicing by hybrid formation at the cappingsite or at the poly A addition site; suppression of translationinitiation by hybrid formation at the binding site for the translationinitiation factors; suppression of translation by hybrid formation atthe site for ribosome binding near the initiation codon; inhibition ofpeptide chain elongation by hybrid formation in the translated region orat the polysome binding sites of mRNA; and suppression of geneexpression by hybrid formation at the sites of interaction betweennucleic acids and proteins. These factors suppress the target geneexpression by inhibiting the process of transcription, splicing, ortranslation (Hirashima and Inoue, “Shin Seikagaku Jikken Koza (NewBiochemistry Experimentation Lectures) 2, Kakusan (Nucleic Acids) IV,Idenshi No Fukusei To Hatsugen (Replication and Expression of Genes),”Nihon Seikagakukai Hen (The Japanese Biochemical Society Ed.), TokyoKagaku Dozin, pp. 319-347, (1993)).

[0050] An antisense sequence used in the present invention can suppressthe target gene expression by any of the above-mentioned mechanisms. Ifan antisense sequence is designed to be complementary to theuntranslated region near the 5′ end of the gene's mRNA; it willeffectively inhibit translation of a gene. Additionally, it is alsopossible to use sequences that are complementary to the coding regionsor to the untranslated regions on the 3′ side. Thus, the antisense DNAused in the present invention includes a DNA having antisense sequencesagainst both the untranslated regions and the translated regions of thegene. The antisense DNA to be used is connected downstream from anappropriate promoter, and, preferably, a sequence containing thetranscription termination signal is connected on the 3′ side. The DNAthus prepared can be transfected into the desired plant by knownmethods. The sequence of the antisense DNA is preferably a sequencecomplementary to the endogenous gene (or the homologue) of the plant tobe transformed or a part thereof, but it need not be perfectlycomplementary so long as it can effectively inhibit the gene expression.The transcribed RNA is preferably not less than 90%, and most preferablynot less than 95% complementary to the transcribed products of thetarget gene. In order to effectively inhibit the expression of thetarget gene by means of an antisense sequence, the antisense DNA shouldbe at least 15 nucleotides long or more, preferably 100 nucleotides longor more, and most preferably 500 nucleotides long or more. The antisenseDNA to be used is generally shorter than 5 kb, and preferably shorterthan 2.5 kb.

[0051] DNA encoding ribozymes can also be used to suppress theexpression of endogenous genes. A ribozyme is defined as an RNA moleculethat has catalytic activities. Numerous ribozymes are known in theliterature, each having distinct catalytic activity. Research on theribozymes as RNA-cleaving enzymes has enabled the designing of aribozyme that site-specifically cleaves RNA. While some ribozymes of thegroup I intron type or the M1RNA contained in RNaseP consist of 400nucleotides or more, others belonging to the hammerhead type or thehairpin type have an activity domain of about 40 nucleotides (Koizumi,Makoto and Ohtsuka, Eiko (1990). Tanpakushitsu Kakusan Kohso (Protein,Nucleic acid, and Enzyme) 35, 2191).

[0052] The self-cleavage domain of a hammerhead type ribozyme cleaves atthe 3′ side of C15 sequence G13U14C15. Formation of a nucleotide pairbetween U14 and A at the ninth position is considered important for theribozyme activity. Furthermore, it has been shown that the cleavage alsooccurs when the nucleotide at the 15th position is A or U instead of C(Koizumi, M. et al. (1988). FEBS Lett. 228, 225). If thesubstrate-binding site of the ribozyme is designed to be complementaryto the RNA sequences adjacent to the target site, one can create arestriction-enzyme-like RNA cleaving ribozyme that recognizes thesequence UC, UU, or UA within the target RNA (Koizumi, M. et al. (1988).FEBS Lett. 239, 285; Koizumi, Makoto and Ohtsuka, Eiko (1990).Tanpakushitsu Kakusan Kohso (Protein, Nucleic acid, and Enzyme), 35,2191; Koizumi, M. et al. (1989). Nucleic Acids Res. 17, 7059). Forexample, in the coding region of the OsGA2ox1 gene (SEQ ID NO: 2)isolated by the present inventors, there are pluralities of sites thatcan be used as the ribozyme target.

[0053] The hairpin type ribozyme is also useful in the presentinvention. A hairpin type ribozyme can be found, for example, in theminus strand of the satellite RNA of tobacco ringspot virus (Buzayan, J.M. (1986). Nature 323, 349). This ribozyme has also been shown totarget-specifically cleave RNA (Kikuchi, Y. and Sasaki, N. (1992).Nucleic Acids Res. 19, 6751; Kikuchi, Yo (1992) Kagaku To Seibutsu(Chemistry and Biology) 30, 112).

[0054] The ribozyme designed to cleave the target is fused with apromoter, such as the cauliflower mosaic virus 35S promoter, and with atranscription termination sequence, so that it will be transcribed inplant cells. However, if extra sequences are added to the 5′ end or the3′ end of the transcribed RNA, the ribozyme activity may be lost. Inthis case, one can place an additional trimming ribozyme, whichfunctions in the cis position to perform the trimming on the 5′ or the3′ side of the ribozyme portion, thereby precisely cutting the ribozymeportion from the transcribed RNA containing the ribozyme (Taira, K. etal. (1990). Protein Eng. 3, 733; Dzaianott, A. M. and Bujarski, J. J.(1989). Proc. Natl. Acad. Sci. USA 86, 4823; Grosshands, C. A. and Cech,R. T. (1991). Nucleic Acids Res. 19, 3875; Taira, K. et al. (1991.)Nucleic Acid Res. 19, 5125). Multiple sites within the target gene canbe cleaved by arranging these structural units in tandem to achievegreater effects (Yuyama, N. et al., (1992). Biochem. Biophys. Res.Commun. 186, 1271). By using such ribozymes, it is possible tospecifically cleave the transcription products of the target gene in thepresent invention, thereby suppressing the expression of the gene.

[0055] Endogenous gene expression can also be suppressed byco-suppression through the transformation by DNA having a sequenceidentical or similar to the target gene sequence. “Co-suppression ” asused herein, refers to the phenomenon in which, when a gene having asequence identical or similar to the target endogenous gene sequence isintroduced into plants by transformation, expression of both theintroduced exogenous gene and the target endogenous gene becomessuppressed. Although the detailed mechanism of co-suppression isunknown, it is frequently observed in plants (Curr. Biol. (1997). 7,R793, Curr. Biol. (1996). 6, 810). For example, if one wishes to obtaina plant body in which the gene of the present invention isco-suppressed, the plant in question can be transformed with a DNAvector designed so as to express the gene of the present invention orDNA having a similar sequence. The gene to be used for co-suppressionneed not be completely identical to the target gene. However, it shouldhave preferably 70% or more sequence identity, more preferably 80% ormore sequence identity, and most preferably 90% or more (e.g. 95% ormore) sequence identity.

[0056] The identity of one amino acid sequence or nucleotide sequence toanother can be determined by following the BLAST algorithm by Karlin andAltschl (Proc. Natl. Acad. Sci. USA, (1993). 90, 5873-5877,). Programssuch as BLASTN and BLASTX were developed based on this algorithm(Altschul et al. (1990). J. Mol. Biol.215, 403-410). To analyze anucleotide sequences according to BLASTN based on BLAST, the parametersare set, for example, as score=100 and word length=12. On the otherhand, parameters used for the analysis of amino acid sequences by theBLASTX based on BLAST include, for example, score=50 and word length=3.Default parameters of each program are used when using BLAST and GappedBLAST programs. Specific techniques for such analysis are known in theart (http://www.ncbi.nlm.nih.gov.)

[0057] Modification of plant growth utilizing a DNA functioning tosuppress the DNA encoding GA 2β-hydrosylase of this invention or itsexpression may be achieved by inserting the DNA into an appropriatevector, transferring the vector into plant cells, and regenerating thetransformed plant cells thus obtained. There is no particular limitationon the type of vectors so far as they are capable of expressing theinserted gene within plant cells. A vector having a promoter (forexample, 35S promoter of cauliflower mosaic virus) that enables theconstitutive gene expression in plant cells may also be used.Furthermore, plant tissue-specific promoters may specifically modifyparticular plant tissues, for example, leaves, flowers, fruits, etc.Examples of the tissue-specific promoters are seed-specific promoterssuch as promoters for β-phaseolin of kidney bean (Bustos, et al. (1991).EMBO J. 10, 1469-1479) and glycinin of soy bean (Lelievre, et al.(1992). Plant Physiol. 98, 387-391); leaf-specific promoters such aspromoters for the RbcS gene of pea (Lam and Chua (1990). Science 248,471-474) and Cab 1 gene of wheat (Gotorn, et al. (1993). Plant J. 3,509-518), root-specific promoters such as promoters for the TobRB7 geneof tobacco (Yamamoto, et al. (1991). Plant Cell 3, 371-382) and rolDgene of Agrobacterium rhizogenes (Elmayan and Tepfer (1995). TransgenicRes. 4, 388-396). It is also possible to use a vector having a promoterinducibly activated by exogenous stimuli. Plant cells into which vectorsare inserted are preferably derived from the same plants as those fromwhich transgenes are derived. However, they are not limited thereto. Infact, the present inventors demonstrated that tobacco plants into whichthe genes derived from Arabidopsis have been introduced also becomedwarfed. Herein, the term “plant cells” includes plant cells in avariety of forms, for example, cultured cell suspension, protoplasts,leaf sections, cali, etc. A vector can be transferred into plant cellsby a variety of methods well known to those skilled in the art,including the polyethylene glycol method, electroporation method,Agrobacterium-mediated method, particle gun method, etc. Regeneration ofa plant body from transformed plant cells may be performed by thestandard methods known in the art. Once the transformed plant body isgenerated, it is also possible to obtain propagative materials (forexample, seeds, tubers, cuttings, etc.) from the plant body and producethe transformed plant of this invention on a large scale.

Brief Description of the Drawings

[0058]FIG. 1, A shows the general structure of gibberellin(ent-gibberellan backborn), and B shows the major GA biosyntheticpathway in higher plants.

[0059]FIG. 2 shows the result of sequence analysis of OsGA2ox1. Adepicts an alignment of the deduced amino acid sequences of2β-hydroxylases from rice (OsGA2ox1), Arabidopsis (AtGA2ox1, AtGA2ox2,and AtGA2ox3), scarlet runner bean (PcGA2ox1 ), and garden pea (PsGA2ox1and PsGA2ox2). B depicts the phylogenetic relationships among GA2β-hydroxylases.

[0060]FIG. 3 shows the metabolic pathway mediated by the recombinantOsGA2ox1 protein. The recombinant OsGA2ox1 protein was incubated withtritium labeled GA₁, GA₄, GA₉, GA₂₀, GA₄₄, and GA₅₃. The products wereseparated by HPLC and identified by GC/MS.

[0061]FIG. 4 is a photograph representing the result of the RNA gel blotanalysis of OsGA2ox1 expression in various organs of a wild-type rice.Total RNA was extracted from vegetative shoot apices (lane 1), youngleaves (lane 2), stems (lane 3), leaf blades (lane 4), leaf sheath (lane5), root (lane 6), inflorescence shoot apices (lane 7), glumes (lane 8),and rachis (lane 9), and hybridized with an OsGA2ox1 cDNA fragment.

[0062]FIG. 5 is a photograph representing in situ mRNA localization ingerminating rice seeds and vegetative shoot apical meristems. Purplestaining indicates the presence of OsGA2ox1 mRNA.

[0063] A: Median longitudinal section of a rice embryo at 3 days aftersowing.

[0064] B: High magnification around a shoot apical meristem.

[0065] C: High magnification around an epithelium and aleurone layer.

[0066] D: Median longitudinal section of a rice embryo stained with asense RNA probe (control).

[0067] E: The longitudinal section of a rice vegetative shoot apicalmeristem. Lines 1, 2, 3, and 4 indicate approximate planes of the crosssections shown in panels F, G, H, and I, respectively. F, G, H, and Iare sequential cross sections of a rice vegetative shoot apical meristemin panel E.

[0068]FIG. 6 is a photograph representing phenotypes of transgenic riceplants overexpressing the OsGA2ox1 cDNA.

[0069] A: Gross morphology of wild-type rice (c) and Act::OsGA2ox1 (1 to5) transgenic rice in a vegetative phase.

[0070] B: Wild-type rice plant at flowering.

[0071] C: Mild dwarf phenotype of Act::OsGA2ox1 transgenic rice.

[0072] D: Moderate dwarf phenotype of Act::OsGA2ox1 transgenic rice.

[0073] E: Severe dwarf phenotype of Act::OsGA2ox1 transgenic rice. Bars.in (B) to (E) represent 10 cm.

Best Mode for Carrying out the Invention

[0074] The present invention will be explained in detail below withreference to Examples, but is not to be construed as being limitedthereto.

[0075] For preparing plant materials used in Examples, rice seeds (Oryzasativa L., Japonica cv. Nippon-bare) were sterilized in 1% NaClO for 1hr and sown on an agar medium. Seedlings were grown in a greenhouse at30° C. (day) and 24° C. (night).

[0076] In the Examples, nucleotide sequences were determined by adideoxynucleotide chain-termination method using an automated sequencingsystem (ABI377). The cDNA and genomic clones were completely sequencedon both strands including a large intron. Analysis of cDNA and aminoacid sequences were carried out using LASERGENE computer software(DNASTAR, Inc., Madison, Wis.). EXAMPLE 1

Isolation of GA 2β-hydroxylase Gene from Rice

[0077] To amplify genomic DNA from rice (Oryza sativa L.) japonica cv.Nihon-bare, two degenerate oligonucleotide primers were designed fromthe conserved region of putative Arabidopsis GA 2β-hydroxylase gene(AtGA2ox3) (cDNA corresponding to DDBJ accession number C72618), Marahmacrocarpa mRNA for dioxygenase (accession number Y09113; MacMillan, J.et al. (1997). Plant Physiol., 113, 1369-1377), rice GA 20-oxidase gene(accession number U50333; Toyomasu, T. et al. (1997). Physiol. Plant.,99, 111-118), rice GA 3β-hydroxylase genes, and other2-oxoglutarate-dependent dioxygenase genes (forward primer,5′-GGNTTYGGNGARCAYWCNGAYCC-3′/SEQ ID NO: 3; and reverse primer,5′-GGISHISCRAARTADATIRTISWIA-3′/SEQ ID NO: 4). PCR was performed usingrice genomic DNA as a template. The amplified fragments (about 80 bp)were cloned into pCR II (Invitrogen, Carlsbad, Calif.) and theirsequences were confirmed. One of the 64 independent clones contained anovel 2-oxoglutarate-dependent dioxygenase-like amino acids and waspredicted to encode a rice GA 2β-hydroxylase gene. This partial aminoacid sequence was used to search the DDBJ Nucleotide Sequence Databaseand one rice EST clone (accession number C72618) was obtained, which ispresumed to encode a rice GA 2β-hydroxylase gene and derived from ear atflowering. Oligonucleotide primers were designed based on the sequenceof this EST clone (forward primer, 5′-GCGGCGTTCTTCGCG-3′/SEQ ID NO: 5;and reverse primer, 5′-CTATTGTGAATGAGTACATT-3′/SEQ ID NO: 6) and used inPCR with rice genomic DNA as a template. The amplified fragments werecloned into pCR II (Invitrogen, Carlsbad, Calif.) and the sequences wereconfirmed. The 230 bp fragment was used as a probe for further screeningfor cDNA and genomic clones.

[0078] Specifically, a cDNA library constructed from rice immature seedmRNA and a genomic library constructed from rice genomic DNA digestedpartially with Sau3AI were screened using a probe prepared as mentionedabove. Hybridization was performed in 5×SSC (1×SSC is 0.15 M NaCl, 15 mMsodium citrate), 5×Denhardt's solution (1×Denhardt's solution comprises0.02% Ficoll, 0.02% PVP, and 0.02% BSA), 0.5% [w/v] SDS, and 20 mg/Lsalmon sperm DNA at 65° C. for 14 hr and filters were washed in 2×SSC,0.1% [w/v] SDS at room temperature.

[0079] The cDNA thus obtained was designated “OsGA2ox1” and contained anopen reading frame of 1,146 bp encoding a protein of 382 amino acids(SEQ ID NO: 2). It contains the sequences that are conserved withindioxygenases of GA biosynthesis, including the His-241, Asp-243, andHis-302 (the numbers refer to the position on OsGA2ox1 amino acidsequence) as mentioned above. An alignment of the amino acid sequencewith those of GA 2β-hydroxylase cDNAs from scarlet runner bean(Phaseolus occineus L.) and Arabidopsis (Thomas, S. G. et al. (1999) ProNatl. Acad. Sci. USA, 96, 4698-4703), and garden pea (Lester et al.(1999) Plant J. 19: 65-73) indicated that OsGA2ox1 is a member of GA2β-hydroxylase (FIG. 2A). However, the phylogenetic relationship amongthese dioxygenases revealed that GA 2β-hydroxylase cDNAs from dicotplants share relatively high (49 to 68%) amino acid identity with eachother, but significantly lower (less than 36%) identity with OsGA2ox1(FIG. 2B).

[0080] The corresponding genomic DNA that completely covers the OsGA2ox1coding region was also cloned. By comparing the genomic DNA sequence andcDNA sequence, it was revealed that OsGA2ox1 comprises three exons andtwo introns. This exon/intron structure is also conserved in theAtGA2ox3 coding sequence.

EXAMPLE 2 Function of Recombinant GA 2β-Hydroxylases

[0081] The full-length cDNA of rice GA 2β-hydroxylase was inserted inthe sense orientation as a translational fusion into the pMAL-c2expression vector (New England Biolabs, Beverly, Mass.). The resultingconstruct, pMAL-OsGA2ox1, was expressed in Escherichia coli strainJM109. Bacterial cells were grown overnight at 37° C. in 2×YT mediumcontaining 0.2% [w/v] glucose and 100 mg/L ampicillin. After overnightgrowth, cultures were diluted 500-fold with the fresh medium andincubated with shaking at 37° C. When growth reached an OD₆₀₀ of 0.7,IPTG was added to a final concentration of 1 mM, and culturing wasresumed at 17° C. for a period of further 24 hr. These bacterial cellswere harvested, washed with washing buffer (50 mM Tris, pH 8.0, 10%[w/v] glycerol, 2 mM DTT), resuspended in the washing buffer containing1 g/L lysozyme, and kept on ice for 30 min.

[0082] The lysate thus obtained was sonicated and centrifuged. Itssupernatant was subjected to SDS-PAGE and the expression of the fusionprotein was confirmed. The supernatant was incubated with ²H-labeled GAsubstrates comprising various C₁₉-GAs, including GA₁, GA₄, GA₉, andGA₂₀Products were identified by GC/MS. Each of the C₁₉-GAs was convertedto the corresponding 2β-hydroxy product by the action of the OsGA2ox1protein (FIG. 3), except for GA₁₉ and GA₅₃ (both of which have openlactone form).

EXAMPLE 3 Expression of GA 2β-Hydroxylase Gene in Rice

[0083] (1) RNA Gel Blot Analysis

[0084] Total RNAs from rice were separately prepared from varioustissues (vegetative shoot apices, young leaves, stems, leaf blades, leafsheath, root, inflorescence shoot apices, glumes, and rachis) for RNAgel blot analysis. Ten μg of each RNA preparation was electrophoresed ona 1.2% agarose gel, transferred onto Hybond N+ membrane (Amersham,Buckinghamshire, England), and then hybridized with the HindIII-EcoRVfragment (the 230 bp fragment of OsGA2ox1 cDNA) as a probe.Hybridization was performed in 5×SSC, 5×Denhardt's solution, 0.5% [w/v]SDS, and 20 mg/L salmon sperm DNA at 65° C. for 14 hr. The filter waswashed in 2×SSC, 0.1% [w/v] SDS at 65° C. and then further washed in0.2×SSC, 0.1% [w/v] SDS at 65° C.

[0085] A single strong band was detected in RNA from all organs examined(FIG. 4). The size of the band was ca. 1.6 kb that was almost the samesize as the cDNA clone.

[0086] (2) In situ Hybridization

[0087] To more precisely determine the spatial pattern of OsGA2ox1expression in rice, in situ hybridization was conducted usingdigoxygenin-labeled OsGA2ox1 antisense-strand RNA as a probe. Plantmaterials were fixed in 4% [w/v] paraformaldehyde and 0.25% [w/v]glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4, overnight at 4°C., dehydrated through graded ethanol series and the t-butanol series(Sass, A. E. (1958). Botanical Micro Technique, 3rd ed. Iowa StateUniversity Press.), and finally embedded in Paraplast Plus (SherwoodMedical). Microtome sections (7 to 10 μm thick) were placed on glassslides treated with Vectabond (Vector Laboratory). Hybridization withdigoxigenin-labeled sense or antisense RNA and immunological detectionof the hybridized probe were conducted according to the method describedby Kouchi and Hata (Kouchi, H. and Hata, S. (1993). Mol. Gen. Genet.,238, 106-119).

[0088] The detected expression patterns of OsGA2ox1 in germinating riceseeds are shown in FIG. 5. Purple staining, indicating the presence ofOsGA2ox1 mRNA, was observed in a ring shaped pattern at the leafinsertion point of the shoot apical meristem, epithelium, and aleuronelayer.

[0089]FIG. 5A shows a near-median longitudinal section through the shootapex of germinating rice seed. OsGA2ox1 expression appears as pairs ofsignal on opposite flanks of the shoot apical meristem (FIG. 5B).Spotted expression was also found in the basal region of differentiatedleaf primordia. In other regions of embryo, OsGA2ox1 expression was seenin the outermost layer of scutellum, epithelium, and aleurone layer(FIG. 5C) Control sections, hybridized with a sense-strand RNA probe,showed no signal above background staining (FIG. 5D).

[0090]FIG. 5E shows the longitudinal section of a rice vegetative shootapex. Lines 1, 2, 3, and 4 in panel E indicate approximate planes of thecross sections shown in FIGS. 5F, 4G, 4H, and 4I, respectively. Byoverlaying the signal of OsGA2ox1 expression observed in serial sections(FIGS. 5F, 4G, 4H, and 4I) it was revealed that the spotted expressionof OsGA2ox1 localized around the boundary between the shoot apicalmeristem and the first leaf primordium in the longitudinal section(FIGS. 5B and 4E) is ring shaped. Similarly, spotted expression in thebasal region of differentiated leaf primordia corresponded to thesignals located around the large vascular bundles (FIGS. 5F, 5G, 5H, and5I).

[0091] The leaf insertion point of the shoot apical meristem andepithelium need a high level of bioactive GA₁ for leaf development andexpansion, and so does the aleurone layer for induction of (x-amylasegene expression to hydrolyze the stored starch in the endosperm duringgermination. Therefore, expression of OsGA2ox1 in the basal region ofdifferentiated leaf primordia, epithelium, and aleurone layer isconsidered to play an important role in regulation of the rapidaccumulation of GA₁ in these tissues. Expression of OsGA2ox1 at thissite seemed to deactivate the GA₁ and prevent an outflow of thebioactive GA to outer tissues where the excess GA₁ induces a confusionof growth program.

[0092] OsGA2ox1 is also expressed in the boundary between the shootapical meristem and the first leaf primordium as a ring-shapedexpression pattern. Such an expression pattern indicates that, throughthe regulation of bioactive GA content, OsGA2ox1 may be involved in orrespond to an early pattern forming event that defines the segmentalunits of the plant body designated phytomers as proposed in the possiblefunction of plant homeobox genes (Schneeberger, R. G. et al. (1995).Genes Devel., 9, 2292-2304; Sato, Y. et al. (1998). Plant Mol. Biol.,38, 983-998). Alternatively, OsGA2ox1 expression and resulting decreaseof GA1 content may mark the future internodes in the postembryonicstages of development. This is suggested by the fact that thering-shaped expression of OsGA2ox1 was observed just below the leafinsertion point around the shoot apical meristem, where the internodewould later develop before any visible differentiation of the node orthe internode is recognized.

EXAMPLE 4 Overexpression of Rice GA 2β-Hydroxylase Gene in TransgenicPlants

[0093] To assess the in vivo activity of the OsGA2ox1 gene product andthe feasibility to modify the endogenous content of GAs in transgenicplants by genetic manipulation of GA 2β-hydroxylase gene expression, thecDNA clone was overexpressed in transgenic rice plants.

[0094] The full-length cDNA of rice GA 2β-hydroxylase was excised as anXbaI-EcoRV fragment and inserted between the rice actin promoter and thenopaline synthase (NOS) polyadenylation signal of hygromycin resistantbinary vector pAct-Hm2. This vector was a modification of pBI-H1 (Ohta,S. et al. (1990). Plant Cell Physiol., 31, 805-813) and contains theactin promoter. The resulting construct was named “pAct-OsGA2ox1”.“pAct-GUS”, which was used as a control vector, was constructed byintroduction of β-glucuronidase (GUS) gene between the actin promoterand the NOS terminator of pAct-Hm2.

[0095] The fusion constructs, “pAct-OsGA2ox1 ” and “pAct-GUS” wereintroduced into Agrobacterium tumefaciens strain EHA101 byelectroporation. Agrobacterium-mediated transformation of rice (Oryzasativa L. cv. Nippon-bare) callus was performed according to the methodof Tanaka et al. (Japanese Patent Application No. Hei 11-206922).Transgenic rice plants were selected on media containing 50 mg/Lhygromycin. Hygromycin-resistant plants were transplanted to soil andgrown at 30° C. (day) and 24° C. (night) in a 16 hr light,8 hr darkcycle.

[0096] More than forty independent transgenic rice plants wereregenerated in this experiment. As expected, all transformantsoverexpressing the OsGA2ox1 cDNA showed dwarf phenotype (FIG. 6). Incontrast to the p35S::AtGA2ox3 transgenic tobacco plants, various riceplants transformed with the pAct::OsGA2ox1 did not show identicalphenotypes but had a range of inhibition of stem elongation (FIG. 6A), aphenomenon caused mainly by various levels of transgene expression indifferent transformants. The mildly dwarfed plants grew up toapproximately 50 cm (FIG. 6C), while the severely dwarfed plants wereless than 15 cm at their final height (FIG. 6E), which was about halfthe height of the wild-type rice (FIG. 6B). The stature of othertransgenic rice plants was varied within this range (FIG. 6D). Thelength of leaf blades was also reduced correlatively with the dwarfstature.

Industrial Applicability

[0097] The present invention has provided novel enzymes and genesinvolved in the inactivation of plant gibberellins as well as plantswhose gibberellin activity has been modified by controlling theexpression of these genes. This invention enables modification ofgibberellin activation in plants so as to artificially modify the planttypes. Inactivation of gibberellin within plants induces plant dwarfphenotypes due to suppression of longitudinal growth. For example, thiscould prevent rice plants from bending over when excessive elongation ispromoted by ample fertilization. A substantial increase in crops mayalso be expected due to enhanced efficiency of light reception toleaves. It is also possible to improve efficiency in harvesting andbreeding management. Another result of the present invention is toincrease the yield of the plant as a whole by suppressing the expressionof genes of this invention in the plant so as to promote gibberellinactivation therein. This strategy is particularly beneficial inimproving the yield of feed crops as a whole.

1 6 1 382 PRT Oryza sativa 1 Met Val Val Pro Ser Ala Thr Thr Pro Ala ArgGln Glu Thr Val Val 1 5 10 15 Ala Ala Ala Pro Pro Ala Ala Ala Ala SerGly Val Val Gly Gly Gly 20 25 30 Gly Gly Val Thr Ile Ala Thr Val Asp MetSer Ala Glu Arg Gly Ala 35 40 45 Val Ala Arg Gln Val Ala Thr Ala Cys AlaAla His Gly Phe Phe Arg 50 55 60 Cys Val Gly His Gly Val Pro Ala Ala AlaPro Val Ala Ala Arg Leu 65 70 75 80 Asp Ala Ala Thr Ala Ala Phe Phe AlaMet Ala Pro Ala Glu Lys Gln 85 90 95 Arg Ala Gly Pro Ala Ser Pro Leu GlyTyr Gly Cys Arg Ser Ile Gly 100 105 110 Phe Asn Gly Asp Val Gly Glu LeuGlu Tyr Leu Leu Leu His Ala Asn 115 120 125 Pro Ala Ala Val Ala His ArgAla Arg Thr Ile Asp Ala Met Asp Pro 130 135 140 Ser Arg Phe Ser Ala IleVal Asn Glu Tyr Ile Glu Ala Met Lys Lys 145 150 155 160 Leu Ala Cys GluIle Leu Asp Leu Leu Gly Glu Gly Leu Gly Leu Lys 165 170 175 Asp Pro ArgTyr Phe Ser Lys Leu Thr Thr Asn Ala Asp Ser Asp Cys 180 185 190 Leu LeuArg Ile Asn His Tyr Pro Pro Ser Cys Asn Ile His Lys Leu 195 200 205 AspHis Asp Asp Gln Cys Asn Ile Lys Ser Leu Val Ser Thr Lys Ala 210 215 220Ser Asn Gly Gly Asn Leu Met Ala Gly Gly Arg Ile Gly Phe Gly Glu 225 230235 240 His Ser Asp Pro Gln Ile Leu Ser Leu Leu Arg Ala Asn Asp Val Glu245 250 255 Gly Leu Gln Val Phe Val Pro Asp His Glu Gly Lys Glu Met TrpVal 260 265 270 Gln Val Pro Ser Asp Pro Ser Ala Ile Phe Val Asn Val GlyAsp Val 275 280 285 Leu Gln Ala Leu Thr Asn Gly Arg Leu Ile Ser Ile ArgHis Arg Val 290 295 300 Ile Ala Thr Ala Cys Arg Pro Arg Leu Ser Thr IleTyr Phe Ala Ser 305 310 315 320 Pro Pro Leu His Ala Arg Ile Ser Ala LeuPro Glu Thr Ile Thr Ala 325 330 335 Ser Ser Pro Arg Arg Tyr Arg Ser PheThr Trp Ala Glu Tyr Lys Thr 340 345 350 Thr Met Tyr Ser Leu Arg Leu SerHis Ser Arg Leu Glu Leu Phe Lys 355 360 365 Ile Asp Asp Asp Asp Ser AspAsn Ala Ser Glu Gly Lys Ala 370 375 380 2 1562 DNA Oryza sativa CDS(54)..(1199) 2 ggcacgagcc attccggccg cgcattctcc cgctctcgat cgatcgatcgatc atg 56 Met 1 gtg gtg cct tcc gcg acg acg cca gcg agg cag gag acg gtggtg gcg 104 Val Val Pro Ser Ala Thr Thr Pro Ala Arg Gln Glu Thr Val ValAla 5 10 15 gcg gcg ccg cca gct gcg gcg gcg tcc ggt gtc gtc ggc ggc ggcggc 152 Ala Ala Pro Pro Ala Ala Ala Ala Ser Gly Val Val Gly Gly Gly Gly20 25 30 ggc gtg acg ata gcg acg gtg gac atg tcg gcg gag cgc ggc gcg gtg200 Gly Val Thr Ile Ala Thr Val Asp Met Ser Ala Glu Arg Gly Ala Val 3540 45 gcg agg cag gtg gcg acg gcg tgc gcg gcg cac ggg ttc ttc cgg tgc248 Ala Arg Gln Val Ala Thr Ala Cys Ala Ala His Gly Phe Phe Arg Cys 5055 60 65 gtc ggg cac ggc gtg ccg gcg gcg gcg ccc gtc gcg gcg agg ctg gac296 Val Gly His Gly Val Pro Ala Ala Ala Pro Val Ala Ala Arg Leu Asp 7075 80 gcc gcg acg gcg gcg ttc ttc gcg atg gcg ccg gcg gag aag cag cgc344 Ala Ala Thr Ala Ala Phe Phe Ala Met Ala Pro Ala Glu Lys Gln Arg 8590 95 gcc ggg ccg gcg agc ccg ctc ggg tac ggc tgc cgg agc atc ggg ttc392 Ala Gly Pro Ala Ser Pro Leu Gly Tyr Gly Cys Arg Ser Ile Gly Phe 100105 110 aac ggc gac gtc ggc gag ctg gag tac ctg ctc ctc cac gcc aac ccc440 Asn Gly Asp Val Gly Glu Leu Glu Tyr Leu Leu Leu His Ala Asn Pro 115120 125 gcc gcc gtc gcg cac cgg gcc agg acc atc gac gcc atg gac ccc tct488 Ala Ala Val Ala His Arg Ala Arg Thr Ile Asp Ala Met Asp Pro Ser 130135 140 145 cgc ttc agt gct att gtg aat gag tac att gaa gcc atg aag aagctc 536 Arg Phe Ser Ala Ile Val Asn Glu Tyr Ile Glu Ala Met Lys Lys Leu150 155 160 gca tgt gag atc ctg gac ctg tta gga gag ggg cta ggt ctc aaggac 584 Ala Cys Glu Ile Leu Asp Leu Leu Gly Glu Gly Leu Gly Leu Lys Asp165 170 175 ccc aga tac ttc agc aag ctt acc aca aac gct gac agt gac tgcctc 632 Pro Arg Tyr Phe Ser Lys Leu Thr Thr Asn Ala Asp Ser Asp Cys Leu180 185 190 ctg agg atc aac cac tac cct cca tca tgc aac att cac aaa cttgac 680 Leu Arg Ile Asn His Tyr Pro Pro Ser Cys Asn Ile His Lys Leu Asp195 200 205 cat gat gac caa tgc aat atc aag agc ctt gtt agc acc aag gctagc 728 His Asp Asp Gln Cys Asn Ile Lys Ser Leu Val Ser Thr Lys Ala Ser210 215 220 225 aat ggt ggg aat ctg atg gca ggt ggg cgc att ggg ttc ggcgag cac 776 Asn Gly Gly Asn Leu Met Ala Gly Gly Arg Ile Gly Phe Gly GluHis 230 235 240 tct gac ccg cag atc ctt agc ttg ctc cga gca aac gat gtggaa ggg 824 Ser Asp Pro Gln Ile Leu Ser Leu Leu Arg Ala Asn Asp Val GluGly 245 250 255 cta cag gtg ttt gtg ccg gac cac gag ggc aag gag atg tgggtt cag 872 Leu Gln Val Phe Val Pro Asp His Glu Gly Lys Glu Met Trp ValGln 260 265 270 gtg cca tcg gac cca tcg gcc att ttc gtc aat gtt ggt gatgtc ctc 920 Val Pro Ser Asp Pro Ser Ala Ile Phe Val Asn Val Gly Asp ValLeu 275 280 285 cag gct ctg aca aat ggg agg ctg ata agt atc cgg cac agggta att 968 Gln Ala Leu Thr Asn Gly Arg Leu Ile Ser Ile Arg His Arg ValIle 290 295 300 305 gca acc gcc tgc agg cca agg ctg tcc aca ata tac ttcgca tca cca 1016 Ala Thr Ala Cys Arg Pro Arg Leu Ser Thr Ile Tyr Phe AlaSer Pro 310 315 320 ccc ctg cat gca cga atc tcg gca ctc cca gag aca atcaca gcc agc 1064 Pro Leu His Ala Arg Ile Ser Ala Leu Pro Glu Thr Ile ThrAla Ser 325 330 335 agc cca cgc cga tac cga tca ttc acc tgg gct gag tacaag acg aca 1112 Ser Pro Arg Arg Tyr Arg Ser Phe Thr Trp Ala Glu Tyr LysThr Thr 340 345 350 atg tac tca ctc cgc ctg agc cac agc cgc cta gaa ctcttc aaa att 1160 Met Tyr Ser Leu Arg Leu Ser His Ser Arg Leu Glu Leu PheLys Ile 355 360 365 gac gat gat gac agc gac aat gcc agt gag gga aaa gcataggaattgc 1209 Asp Asp Asp Asp Ser Asp Asn Ala Ser Glu Gly Lys Ala 370375 380 tggttaaatt gcagacgatg cctatggacc agtggggatt aggaagctgaaactgtcccc 1269 aaaattttgg ctctctggca gtctggctac tatcgtcaga tatctcactattatgatggt 1329 gtagtgccta agttgacggg tgtgtaatat cgttagcagt ctacagaagctatggttgta 1389 cggaagtaat gtactgtcgc cttttcagct aactatccat gttctctcttatatgtaatg 1449 agttagttga cggatgtgta atattgctag cattgtatat aagctatggttgtatggaag 1509 tatgtaatat agccttttca gctaaaaaaa aaaaaaaaaa aaaaaaaaaaaaa 1562 3 23 DNA Artificial Sequence Description of ArtificialSequencean artificially synthesized primer sequence 3 ggnttyggngarcaywcnga ycc 23 4 25 DNA Artificial Sequence Description of ArtificialSequence an artificially synthesized primer sequence 4 ggnshnscraartadatnrt nswna 25 5 15 DNA Artificial Sequence Description ofArtificial Sequencean artificially synthesized primer sequence 5gcggcgttct tcgcg 15 6 20 DNA Artificial Sequence Description ofArtificial Sequencean artificially synthesized primer sequence 6ctattgtgaa tgagtacatt 20

1. A DNA encoding a protein having gibberellin 2β-hydroxylase activity,selected from the group consisting of: (a) a DNA encoding a proteincomprising the amino acid sequence set forth in SEQ ID NO: 1, (b) a DNAcontaining a coding region of the nucleotide sequence set forth in SEQID NO: 2, and (c) a DNA encoding a protein comprising the amino acidsequence set forth in SEQ ID NO: 1, wherein one or more amino acidresidues are substituted, deleted, added, and/or inserted.
 2. A DNAaccording to claim 1, which is used for producing dwarfed plants.
 3. ADNA for suppressing the expression of endogenous DNA according to claim1 within plant cells, selected from the group consisting of: (a) a DNAencoding an antisense RNA complementary to the DNA according to claim 1or its transcription product, (b) a DNA encoding an RNA having theribozyme activity to specifically cleave the transcription product ofthe DNA according to claim 1, and (c) a DNA encoding an RNA thatsuppresses the expression of the endogenous DNA according to claim 1 byco-suppression, wherein said DNA has 70% or more homology to a DNAcomprising the nucleotide sequence set forth in SEQ ID NO:
 2. 4. Avector harboring the DNA according to any one of claims 1 through
 3. 5.A transformed plant cell harboring the DNA according to any one ofclaims 1 through 3 in an expressible state.
 6. A transgenic plantcontaining the transformed plant cell according to claim
 5. 7. Apropagative material of the transgenic plant according to claim
 6. 8. Aprotein encoded by the DNA according to claim
 1. 9. A method forproducing the protein according to claim 8, wherein said methodcomprises culturing the transformed cells harboring the DNA according toclaim 1 in an expressible state and recovering the expressed proteinfrom said cells or the culture supernatant thereof.
 10. A method formodifying the plant growth, wherein said method comprises controllingthe expression level of the DNA according to claim 1 in plant cells. 11.A method for modifying a plant type, wherein said method comprisescontrolling the expression level of the DNA according to claim 1 inplant cells.
 12. A method according to claim 10 or 11, wherein the DNAaccording to any one of claims 1 through 3 is expressed in plant cells.